High-density arrays (HDAs) are new tools used by drug researchers and geneticists to provide information on the expression of genes. A high-density array typically comprises between 5,000 and 50,000 probes in the form of single stranded DNA, each of a known and a different sequence, arranged in a predetermined pattern on a substrate. The arrays are used to test whether single stranded target DNA sequences interact or hybridize with any of the single stranded probes on the array. The testing procedure consists of printing and binding single-stranded DNA molecules onto a substrate. The substrate may be any size, but typically takes the form of a standard 1×3 inch microscope slide. The printed DNA sequence is for a known genetic risk factor and may be tagged with a fluorescent marker for identification. Unknown, single-stranded DNA, such as obtained from a patient, is tagged with a different fluorescent marker and washed over the slide for a specified period of time and then rinsed. If the unknown DNA contains any strands that have complementary nucleic acid sequences to the known strand, hybridization occurs. Any hybridization on the rinsed slide is detected as fluorescence from the marker on the unknown DNA. Fluorescence above a predetermined, threshold intensity indicates that the unknown DNA contains that risk factor associated with the known DNA printed on the slide.
After exposing the array to target sequences under selected test conditions, scanning devices can examine each location on the array and determine the quantity of targets that are bond to complementary probes. The ratio of fluorescent intensity at each spot on the high-density array provides the relative differential expression for a particular gene. DNA arrays can be used to study the regulatory activity of genes, wherein certain genes are turned on or “up-regulated” and other genes are turned off or “down-regulated.” So, for example, a researcher can compare a normal colon cell with a malignant colon cell and thereby determine which genes are being expressed or not expressed in the aberrant cell. The regulatory cites of genes serves as key targets for drug therapy.
Proper performance of a DNA array depends on two basic factors: 1) retention of the immobilized probe nucleic sequences on the substrate, and 2) hybridization of the target sequence to the immobilized probe sequence, as measured by fluorescence emission from the tagged target sequence. The DNA probe material must be retained on the surface of the substrate through a series of washing, blocking, hybridizing, and rinsing operations that are commonplace in DNA hybridization assays. An excessive loss of probe DNA sequences can lead to a low fluorescent-signal-to noise ratio and uncertain or erroneous results.
DNA arrays have for years been printed onto organic, micro-porous membranes such as nylon or nitrocellulose. The densities at which one can print DNA solutions onto these types of organic micro-porous membranes is limited because of the tendency for the DNA solution to wick laterally through the membrane, thus causing cross-talk and contamination between adjacent locations. Others have employed a flat, non-porous substrate surface made from glass. (See for example, U.S. Pat. No. 5,744,305, incorporated herein by reference.) These substrates, however, have also been found wanting, since they do not retain the probe molecules as well as porous substrates.
The present invention proposes to use a substantially flat, porous, inorganic substrate surface that is specially treated with cationic polymer coating to enhance retention of nucleic moieties for high-density arrays. The porous surface provides increased surface area for immobilizing DNA probe molecules, which increases the density of DNA binding sites per unit cross-sectional area of the substrate. The increased number of possible binding sites per unit area results in greater retention of immobilized DNA probes and the emission of an increased signal when hybridized with target molecules.